Directed DNA Polymerase Evolution: Effects of Mutations in Motif C on the Mismatch-Extension Selectivity of Thermus aquaticus DNA Polymerase

• Published in: Chembiochem : a European journal of chemical biology Vol. 8; no. 4; pp. 395 - 401
• Main Authors: Strerath, Michael, Gloeckner, Christian, Liu, Dan, Schnur, Andreas, Marx, Andreas
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 05.03.2007; WILEY‐VCH Verlag
• Subjects: Amino Acid Motifs; Amino Acids - chemistry; Base Sequence; directed evolution; DNA; DNA - chemistry; DNA - genetics; DNA polymerase; Evolution, Molecular; Molecular Sequence Data; Mutation - genetics; Polymerase Chain Reaction; single nucleotide polymorphism; Taq Polymerase - chemistry; Taq Polymerase - genetics; Thermus - enzymology
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.200600337

The selectivity of DNA polymerases for processing the canonical nucleotide and DNA substrate in favor of the noncanonical ones is the key to the integrity of the genome of every living species and to many biotechnological applications. The inborn ability of most DNA polymerases to abort efficient extension of mismatched DNA substrates adds to the overall DNA polymerase selectivity. DNA polymerases have been grouped into families according to their sequence. Within family A DNA polymerases, six motifs that come into contact with the substrates and form the active site have been discovered to be evolutionary highly conserved. Here we present results obtained from amino acid randomization within one motif, motif C, of thermostable Thermus aquaticus DNA polymerase. We have identified several distinct mutation patterns that increase the selectivity of mismatch extension. These results might lead to direct applications such as allele‐specific PCR, as demonstrated by real‐time PCR experiments and add to our understanding of DNA polymerase selectivity. Fussy, fussy. Mutant DNA polymerases with greater discrimination against the extension of mismatched substrates have been identified through the randomization and screening of the Gln‐Val‐His sequence in motif C of T. aquaticus polymerase (see graphic).